This R21 exploratory proposal uses an integrated approach to discover lead candidates suitable for further lead optimization as therapeutics for chronic hepatitis B virus (HBV) infections. Our long-term objective is to develop small molecule inhibitors of HBV biosynthesis with a novel mechanism of action distinct from the currently available drugs. A broad consensus exists in the HBV research community that the key to the successful development of new novel classes of HBV medications lies in targeting steps in the viral life cycle other than replication mediated by the HBV polymerase/reverse transcriptase. Currently available medications, including type 1 interferon and nucleosides analogs, do not offer a significant probability for the resolution of the chronic carrier state. The shortcomings o the existing HBV therapeutic regiments include the necessity of life-long treatment and suboptimal patient acceptance and adherence. Clearly, new drugs with novel mechanisms of action are needed to find a cure leading to complete eradication of HBV infection. Antiviral therapy development targeting not viral, but cellular components essential for viral biosynthesis may represent a novel approach for the generation of HBV therapeutics. A combination of nucleoside analogs with the novel drugs targeting host factors may potentially lead to the prevention of new cccDNA synthesis. Recent advances in target identification techniques for therapeutically relevant chemical compounds and the availability of simple assays amenable to HTS analysis for the evaluation of HBV transcription allow for a multitude of methods to discover new therapeutic agents for HBV. We hypothesize that it will be possible to identify and design inhibitors of HBV biosynthesis by specifically targeting cellular factors that are essential for vial pregenomic RNA synthesis using a combination of approaches: high- throughput screening (HTS) against HBV nucleocapsid promoter activity, computer-aided drug design (CADD), chemical biology, target identification methods, target-oriented rational drug design, medicinal chemistry, and evaluation for efficacy of the most promising compounds shown to inhibit HBV transcription and replication in cell culture. To achieve this goal, our specific aims are as follo: Aim 1. Identify lead- or drug-like low molecular weight inhibitors of HBV nucleocapsid promoter activity using high- throughput screening of chemical libraries with available primary HTS, counter-screening HTS, and secondary viral replication assays. Aim 2. Design and synthesize analogs of the most promising HBV antiviral compounds from aim 1 to generate a SAR and identify compounds suitable for cell-based and photoaffinity-based target identification experiments. If time permits, pilot target identification studies using non-hepatoma cells complemented with a panel of nuclear receptors capable of supporting HBV transcription and replication, and photoaffinity labeling followed by LC-MS/MS analysis will be performed.